JP2003245097A - Gene expression for analyzing light injury - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a personal care method for skin that can detect the light injury. <P>SOLUTION: The first gene expression of a skin sample having known skin conditions and the second gene expression of a skin sample having no known skin condition are measured. The expressions of the genes are compared and evaluated by using a plurality of light injury markers. They are provided to a polynucleotide sequence as a light injury marker. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a polynucleotide sequence functioning as a marker for photodamage in a gene array, and a method for detecting photodamage using these markers.

[0002]

BACKGROUND OF THE INVENTION This application was filed on Nov. 8, 2001, and is US Provisional Application No. 6 incorporated herein by reference.
No. 0 / 337,856 to US Patent Act 119
Make a claim based on the article.

All the genes of a cell make up the genome. The human genome contains approximately 40,000 genes. However, only a fraction of these genes are expressed or phenotypically expressed in any given cell. Phenotype refers to the visible characteristic of an organism that results from the interaction of genotype with the environment. Therefore, in each cell type only a small part of the human genome is expressed at any given moment.
The timing of expression of each gene is accurate, and the level is also accurate.

Automated DNA sequencers have facilitated the sequencing of the organism's genome. For example, Haemo
philius influenzae, Mycopl
asma genitalium, and Caenor
Habditis elegans genomic sequences have been published, which offers the possibility of obtaining genomic sequences of higher organisms such as humans (Fl
eischmann, R.A. D. et al. (199
5) Science 269: 496; Fraser,
C. M. et al. (1995) Science 2
70: 397; Hodgkin, J .; et al. (1
995) Science 270: 410).

A given mammalian cell of a given lineage expresses about 20,000 to 30,000 of the 40,000+ germline genes contained in its genome. Almost all cells ubiquitously express many genes, which are called "housekeeping" genes.
Examples of housekeeping genes include genes encoding enzymes involved in glycolysis or proteins involved in cell structure. However, it is the non-universally expressed genes that differentiate cells. As a cell matures into a differentiated cell, certain non-constantly expressed genes turn on and off at different stages. Therefore, for example, a nerve cell becomes different from a blood cell due to a difference in gene expression pattern between cells.

In abnormal cell conditions, such as conditions in individuals with a disease or disorder, the pattern of gene expression within individual cells is altered as compared to the expression pattern seen in normal, non-diseased conditions. there is a possibility. Altered gene expression may be the result or cause of a disease or disorder, eg in tumor cells. Although some diseases are understood to be caused by mutations in specific genes, they can potentially be detected by examining their genomic sequences, but many diseases and disorders are associated with gene expression levels. With dysfunction in
It cannot be detected by sequencing the genome, but only by identifying the gene expression pattern of the cell. Therefore, to understand the function of a particular cell type in an organism (at a particular time during its life), or to understand the progress of a disease or disorder, at different stages of development of that organism. It is necessary to understand the expression status of individual genes within these particular cell types.

Skin aging is believed to consist of two simultaneous processes. The first process is endogenous, aging over time, probably similar to the aging of other tissues (Uitto, 1986). The second process is photoaging, a remodeling of the dermis caused by the environment that results from repeated exposure of the skin to sunlight. Recent studies (Varani et al.,
1998; Varani et al., 2000) that both intrinsic and photoaging have certain common properties, such as decreased procollagen gene expression and increased expression of genes encoding several matrix metalloproteinases. And it is suggested that photoaging is a major factor contributing to prematurely aged appearance of the skin exposed to sunlight (Yaa).
r and Gilchrest, 1998).

Clinically, sun-damaged skin is characterized by wrinkles, loss of elasticity, and altered texture (Kligman, 1989; Taylor et al., 19).
90). In his previous studies, these features were primarily attributed to changes in the dermis, as histopathological analysis revealed alterations of various extracellular matrix proteins in the dermis of skin exposed to sunlight. I was told. The most prominent of these changes in the dermis are the superficial dermis of the sun-exposed skin.
It is obvious that elastic fibers with morphological changes are significantly accumulated. This accumulation of abnormal dermal elastic fibers following exposure to sunlight results in solar elastosis (solar e
(lastosis) (Gilchres
t, 1989).

[0009] The cellular mechanism that causes sunlight-induced elastic fibrosis is not understood, and on the contrary, controversial findings have been reported regarding the synthesis of elastic fibers during sunlight-induced elastic fibrosis. In some reports, the elastic fibers that accumulate during actinic elastic fibrosis consist of the same components as normal elastic fibers, which are called elastin, a cross-linked insoluble protein that makes up the amorphous component of elastic fibers. , And fibrillin, a major component of elastic fiber microfibrils. Keratinocytes are U
In response to VA and / or UVB radiation, it secretes a number of mediators capable of stimulating fibroblast synthetic activity, some of which, eg TGF-.
β, IL-1β, and IL-10 have been shown to increase elastin gene promoter activity, steady-state mRNA levels, and increased elastin accumulation (Kahari et al., 1992; Mauviel et al., 1).
993; Reitamo et al., 1994). Bernst
Ein et al. (1994) reported increased elastin mRNA levels in sun-damaged skin, but Werth et al. (Werth et al., 1997).
Report that there is no difference in the steady-state level of elastin mRNA during actinic elastic fibrosis. The latter finding suggests that a post-transcriptional mechanism leads to enhanced translational efficiency responsible for elastin accumulation in the absence of elevated mRNA levels, in response to UV irradiation (Schwartz et al., 1995). Is consistent with the study. These results imply that aberrant expression of genes encoding proteins that make up elastic fibers as a result of UV exposure may be the basis of solar elastic fibrosis. Moreover, in some reports,
Steady-state mRN of fibrillin as well as elastin
Changes in A level have been demonstrated (Bernstein
1994). Additional observations also refer to altered levels of elastic fiber proteins such as lysyl oxidase, a copper-dependent amine oxidase responsible for catalysis of elastin cross-linking (Smith-Mun.
go and Kagan, 1998).

Other changes in extracellular matrix proteins in response to UV irradiation have also been demonstrated. For example, the amount of collagen fibrils has been shown to be dramatically reduced in photoaged skin. It is suggested that this change is not associated with changes in collagen mRNA levels, but that degradation of collagen fibrils is associated with UV exposure (Ber.
Nstein et al., 1996). To explain these changes in collagen deposition, Voorheees et al. Propose that UV irradiation induces increased growth factors and cytokine receptor synthesis in fibroblasts and keratinocytes. This increase in receptor synthesis subsequently activates the transcription factor AP-1 through the MAP kinase (mitogen activated protein kinase) signaling cascade (Fisher et al.
1996; Fisher and Voorhees, 19
98), increased expression of genes encoding several matrix metalloproteinases that degrade collagen (Fisher et al., 1996) and decreased expression of genes encoding type I and type III procollagen.

This model of AP-1 activation of matrix metalloproteinase gene expression is an attractive hypothesis, but many other alterations of extracellular matrix have been shown to be associated with UV exposure. Does not explain. Furthermore, the pathological biology of sun-damaged skin is complicated by multiple direct and indirect changes in gene expression in the dermis and epidermis (AP-1 activation is just one example of such changes). It is likely to appear through interaction.

The complex cascade of events associated with sun damage is poorly understood. To identify alterations in transcript profiles in response to exposure to sunlight, researchers have studied many techniques, including, for example, isolating proteins from different cells and comparing the abundance of each of those proteins. Have used technology. Another way is
It uses antibodies to probe the population of peptides produced from the mRNA pool. Therefore, as described in US Pat. No. 5,242,798, mRN
A "library" of synthetic polypeptides corresponding to the polypeptide encoded by the A molecule is made and then probed with the individual antibodies.

In parallel with the progress made in determining which genes are expressed by a given tissue or cell,
Significant advances have been made in the biotechnology industry in the design and manufacture of gene "array" technology. SAGE (Ser
ial Analysis of Gene Express
techniques such as ssion) can be used to generate data on keratinocytes (or epidermis) and thereby develop gene arrays.

Gene arrays are solid phase systems containing fixed nucleotide sequences representing up to several thousand individual genes of interest (of known or unknown function). Utilizing such arrays, tissue or cell culture extracts are tested to determine which genes are on or off in response to treatment, injury, age, sex, ethnicity, drugs, food, and cosmetics. Can decide. However, with this method available in the prior art, it is still difficult to track the expression of even a few genes in experimental models or in human tissues.

[0015] Several patents relate to the use of SAGE technology, or to the fabrication of arrays, making arrays,
Protects equipment designed for processing. For example, EP 799897 discloses methods and compositions for selecting tag nucleic acids in a probe array. W
O9743450 discloses a hybridization assay on an oligonucleotide array. WO9
815651 discloses a method for identifying antisense oligonucleotide binding. However, none of the known patents disclose the construction and use of specific gene arrays for the identification of photodamage.

As used herein, the term "compr" is included.
"issing)" includes (made up)
of), composed of, and / or consisting essentially of.
Unless stated otherwise in the examples and comparative examples, or unless expressly stated otherwise, all numbers in this description indicating the conditions of the material or reaction, the physical properties of the material, and / or the amount or ratio of use. It is to be understood that is modified by the word "about".

As used herein, the term "skin" refers to
Includes skin on face, neck, chest, back, arms, hands, legs, and scalp. The term "epidermal" or "keratinocyte"
It is considered to be the same as included in the term "skin".

[0018]

[Non-Patent Document 1] Fleischmann, R. et al. D. e
t al. (1995) Science 269: 496.

[Non-Patent Document 2] Fraser, C.I. M. et al.
(1995) Science 270: 397.

[Non-Patent Document 3] Hodgkin, J. et al. et al.
(1995) Science 270: 410.

[Patent Document 1] US Pat. No. 5,242,798

[Patent Document 2] EP 799897

[Patent Document 3] WO9743450

[Patent Document 4] WO9815651

[Patent Document 5] US Pat. No. 5,695,937

[0019]

SUMMARY OF THE INVENTION The present invention relates to a polynucleotide sequence that functions as a marker for photodamage in a gene array, and a method for detecting photodamage using these markers.

[0020]

A personal care method for detecting an optical disorder, comprising: (a) SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 5
9, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 6
6, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69
Using at least one photodamage marker selected from one or more sequences selected from the group consisting of: (b) detecting a change in the marker,
A method comprising the step of determining the presence of a light obstruction.

[0021]

DETAILED DESCRIPTION OF THE INVENTION As used herein, the following terms will be understood.

[Medical App (Medical App
"lication)" are devices and compositions that are distributed only by prescription or to the medical profession.

"Personal care applications" are devices and compositions other than medical applications for cleansing and caring for human skin.

A "gene" is a unit of inheritable genetic material found in human chromosomes.

The repeating constitutional units of all nucleic acids are eight different nucleotides, four kinds of nucleotides are constitutional units of DNA and the other four kinds are constitutional units of RNA. For example, a four letter word for DNA is translated into a twenty letter word for protein.

An "oligonucleotide" is an oligomeric fragment of two or more, preferably more than three deoxyribonucleotides or ribonucleotides.

A "polynucleotide sequence" is a polymeric chain of mononucleotides in a given order. As for the polynucleotide sequence, the 5'to 3'end or its complementary strand, 3'to 5 ', has been reported.

An "expressed sequence tag"("EST") is a nucleotide sequence containing a sufficient number of base pairs, such as one that specifically specifies a cDNA (complementary deoxyribonucleic acid) sequence. ESTs can be isolated or purified.

"Isolated" means nucleic acid separated from other cellular components.

"Purified" means an isolated nucleic acid mixture that has been stripped of other materials as much as possible to leave only the nucleic acid.

Gene expression analysis is a tool that can be used to identify markers that are indicative of a particular skin condition such as photodamage or dry skin. Light disorders, dry skin, oily skin, and other "cosmetic" skin conditions are poorly understood biologically. Traditionally, these conditions have been studied by addressing one biological pathway at a time. The present invention provides an application of SAGE technology, described in more detail below, to comprehensively study skin conditions and elucidate new pathways. The present invention provides polynucleotide sequences that are indicative of a particular skin condition. Specifically, the present invention provides specific "ESTs" (sets of genes) that are altered in photodamage and therefore can be used as markers of photodamage. The EST or marker of the present invention is
It was never known before to be important or used to identify light damage.

A preferred method of identifying a polynucleotide sequence is SAGE (Serial Analysis), as described in US Pat. No. 5,695,937.
by using Sof Gene Expression). This technique allows the analysis of multiple transcripts. In essence, it produces a cDNA oligonucleotide. The first defined nucleotide sequence tag is then isolated from the first cDNA oligonucleotide and the second defined nucleotide sequence tag is isolated from the second cDNA oligonucleotide.
Determining the nucleotide sequences of the first and second tags, these tags correspond to the expressed gene.

The present invention is an ES for confirming optical interference.
Methods of using T and the proteins it encodes are provided. The method selects a first epidermal sample having at least one sequence and a second epidermal sample having the same sequence.
Including a first step of selecting a skin sample of. First
The epidermis sample of is compared to the second epidermis sample to determine if there is a change in sequence. If there is a change in the sequence, there is a photodamage in the second epidermal sample. The same method can be applied to samples of dermis or whole skin.

SAG of the skin behind the auricle and the skin before the auricle
Comparison of E libraries To identify genes that are specifically expressed in sun-exposed skin, SAG was applied to the skin before and after the ear.
The E libraries were compared. A small but significant fraction of the SAGE sequence tags analyzed showed significant differences in pre-auricle and retro-auricular skin copy numbers. In the anterior skin exposed to the sun, 19 unique tags were found at fairly low levels (at least a quarter), whereas in the anterior skin, 15 Individuals were seen at a level at least 4 times higher. Tables 4 and 5 list these tags with significantly different copy numbers. Of these tags, 24 were specifically matched to the UniGene database and 10 were multiply matched or not matched. Three of these non-matching tags have a sequence predominantly composed of multiple deoxyadenosine residues: Tag SEQ ID NO: 51 (CAAA in Table I).
AAAAAA) and tag SEQ ID NO: 65 (GGAAA)
AAAAA), tag SEQ ID NO: 77 in Table II (TAA
AAAAAAAA). Another four tags make a reliable match to two different genes, one tag for any of the oriented GenBank cDNs.
It did not show any significant similarity with the A sequence, but other SAGE
Found in the library, one tag is UniGen
It did not match e or any other SAGE library. The remaining unidentified tags are Alu
Represents a repetitive sequence, resulting in a large number of matches.

Of the 24 tags that were specifically matched, it was observed that the skin of the anterior pinna had a 7-fold higher number of keratin 1 tags. We also found increased copy number for tags derived from several other genes (Table II) in sun-damaged skin. These include the following:

1. The psoriasin gene is S10
A tag encoding a member of the 0 calcium-binding protein family and derived from this gene was found to be at 4-fold higher levels in the anterior skin. Pso
Riasin protein and mRNA levels are
in skin exposed to UVB in vivo 10 after exposure
It has been reported to rise to day (Di Nuzz
o et al., 2000). In addition, Psoriasin has been previously shown to be present in all layers of psoriatic epidermis, has been shown to be associated with epidermal fatty acid binding protein (EFABP), and the gene encoding Psoriasin has also been shown to The gene encoding EFABP is also
It is known to be upregulated in psoriasis (H
agens et al., 1999). EFABP gene expression has also been shown to be induced in human skin by topical application of retinoic acid (Larsen et al.,
1994). According to the SAGE data of the present invention, EFABP mR was higher in the skin exposed to the sun than in the normal skin.
A 6-fold higher number of tags was shown for NA.

2. The mRNA encoding insulin-like growth factor binding protein 6 (IGFBP-6) is represented by 6 tags in the anterior skin and 1 tag in the posterior skin, and IGFBP-6 is It binds insulin-like growth factor II (IGF-II) with high affinity, and this binding suppresses the action of IGF-II. Three groups of IGFBP proteases (matrix metalloproteinase, kallikrein, and cathepsin) have been shown to cleave the IGFBP-IGF complex and release functional IGF from its binding protein. IGFBP-6 was associated with quiescent, non-proliferating cells, which was IGFBP-
6 has been suggested to act as an autocrine growth factor (Kato et al., 199).
5). Kelley et al. (Kelley et al. 1996)
Suggests that IGFBPs also have additional intrinsic biological activity independent of IGF.

3. Calmodulin-like skin protein (C
The mRNA for LSP) is represented by a 16: 4 tag ratio in sun-damaged and sun-protected skin. CLSP is a recently identified protein and is reported to be particularly abundant in the epidermis. Furthermore, C
LSP gene expression has been shown to be directly associated with keratinocyte differentiation (Mehul et al., 200.
0).

4. Macrophage migration inhibitory factor (MI
F) mRNA was upregulated 4-fold in the anterior skin. Originally reported to be released by activated T cells, MIF blocks macrophage migration and activates macrophages at sites of inflammation. Moreover, previous studies have linked MIF as a regulator of epidermal immunity and cell differentiation (Shim).
Izu et al., 1995). UVB irradiation is in vivo
And in vitro, it has been shown to induce MIF production in human epidermal keratinocytes (Shimizu et al., 1999). In addition, MIF is also thought to be involved in psoriasis, as MIF levels are elevated in psoriasis plaques (Stei).
nhoff et al., 1999). Therefore, MIF functions as an inhibitor of anti-inflammatory action by regulating some proinflammatory cytokines and by controlling the immunosuppressive effects of steroids on immune cell activation and cytokine production. It seems that

5. In the anterior skin library of the present invention, a testis enhancing gene (Testis enhance)
Four SAGE tags for the ed gene) transcript (TEGT) were identified, whereas only one tag was detected in all posterior pinna tags. TEG
T is pro-apoptotic
It has been found to be consistent with bax inhibitor 1 (BI-1), a recently described repressor of the protein bax (Xu and Reed, 1998). The mechanism of apoptosis suppression has not been clarified yet, but BI-
1 has been shown to have less effect on the level of bax. Therefore, BI-1 is equal to bax
Indirectly, probably the anti-apoptotic protein bcl
It was suggested to suppress by using it instead of -2.

6. Sergiline (c
The number of tags representing ellugyrin) mRNA increased 4-fold. Sergillin is synaptogylline (syna)
Ptogyrin) is a widely expressed member of the synaptic vesicle protein family and is essential for the regulation of synaptic vesicle transport (Janz and Sudho.
f, 1998). For example, in adipocytes, insulin activates the translocation of membrane vesicles containing glucose transporter 4 (Glut4) from the intracellular compartment to the plasma membrane, ultimately increasing glucose uptake. Since redistribution of Sergillin-positive Glut4 vesicles to the plasma membrane is not initiated by insulin stimulation, these vesicles have specific functional properties independent of Glut4 vesicles that migrate to the plasma membrane. It is thought that they are doing (K
uprianova and Kandror, 200
0).

7. M for imogen 38 (mitochondrial 38 kD islet antigen)
RNA is also represented by a 4-fold increase in tag number in the anterior skin. This antigen is one of the molecular targets of autoreactive T cells in type I diabetes (insulin-dependent diabetes mellitus).

Reduced number tags (Table I) from mRNAs encoding known proteins in sun-exposed skin include:

1. Cathepsin D (SEQ ID NO: 55) was shown to have the most marked (less than one-fifth) reduction in mRNA steady-state levels in the anterior skin. Cathepsin D is a lysosomal aspartate proteinase known to be present in the epidermis as well as many other tissues. The timing of activation and degradation of this protein has been shown to be associated with the stage of cell differentiation, and expression of cathepsin D in the epidermis is associated with changes in calcium concentration, such as keratin 10, involucrin, and transglutaminase. Similar to the expression of other structural proteins that respond (Horikoshi et al., 19
98).

2. A typical SAGE in our anterior auricular library compared to the posterior auricular library
Ladinin mRNA (SEQ ID NO: 5 in Table I below was shown to have a one-fifth reduction in tag.
6) is an anchoring-film.
ent) -related protein and contributes to some autoimmune disorders such as linear IgA disease.
nt) is one of the related proteins (Mol and M
Oll, 1998).

3. SEQ ID NO: 57 in Table I below was found to be 9-fold in the retro-auricular skin and only 2-fold in the pre-auricular skin. This tag has two different UniG
The ene database entries were matched, one for the mRNA encoding lecithin-cholesterol acyltransferase (LCAT) and the second for the mRNA encoding the Bcl-2-antagonist (Bak). LCAT is an important enzyme in converting cholesterol into cholesteryl ester and maintaining homeostasis of cholesterol in blood.
Infections or inflammation disrupt lipoprotein metabolism and plasma concentrations of lipids and lipoproteins, and LCAT is known to change under these conditions (Khovidhunkit et al., 2000).
On the other hand, Bak is a pro-apoptotic protein having high sequence homology with bax. Both proteins
It oligomerizes in the mitochondrial membrane and forms pores that facilitate cytochrome c outflow (Korsmeyer et al., 20
00), it is believed to induce an apoptotic cascade.

4. In the skin damaged by sunlight, a 1/4 mRNA level of mRNA encoding dyxin was detected as compared with normal skin. Dixin is a local adhesive phosphoprotein that has been reported to be expressed in all layers of the epidermis (Leccia et al., 1999). In addition, dyxin also allows proteins to bind to the cell-substrate interadhesive junction (adhere).
ns junction) and fibroblasts, which have been shown to coexist with cell-cell adhesive junctions. Dixin consists of (LIM domain, double (d
possessing structural properties (such as Zn finger motifs) along with signal transducers involved in developmental regulation, and in previous studies dyxin also
It has also been suggested that it may be involved in the regulation of cell proliferation and differentiation (Beckerle, 1997).

5. SAGE libraries derived from sun-damaged skin were shown to have reduced levels of mRNA encoding the calcium ion binding protein S100 A3. Like Psoriasin, S100 A3 is a member of the S100 calcium binding gene family. S100 A3
The significant expression in mouse of the gene encoding
Restricted to the hair follicle, the timing of expression of this gene is at the same time as the neonatal and adolescent phases of the hair growth cycle (Kiza).
wa et al., 1998).

6. Cartilage matrix oligomeric protein (ca
rtilage oligomeric matrix
Another Ca called protein (COMP)
^{For 2+} binding proteins, a decrease in the number of tags in sun-exposed skin was observed. COMP is an extracellular matrix glycoprotein that is expressed not only in cartilage and ligaments, but also in human dermal fibroblasts (Dodge et al., 1998) and human vascular smooth muscle cells (Riessen et al., 2001) in vitro. is there. CO
Mutations in MP reduce calcium binding capacity,
Pseudochondrodysplasia (PSAC) is a skeletal disorder
H) has been shown to occur (Maddox et al.,
2000). However, otherwise, little is known about the function of COMP.

In the skin of the anterior part of the ear exposed to sunlight, a tag (ACATCATCG) derived from ribosomal RNA was used.
AT-SEQ ID NO: 53 and ACTCCAAAAAA-SEQ ID NO: 54), as well as tags derived from mRNAs encoding unknown proteins (CAAAAAA).
AAAA-SEQ ID NO: 51 and ACGTTAAAGA-
A reduced number of SEQ ID NO: 52) was observed.

[0051]

[Table 1]

[0052]

[Table 2]

Example 1 To study phenotypic changes at the transcriptome level in human skin repeatedly exposed to sunlight, the skin of the anterior part of the ear damaged by sunlight and the ear protected from sunlight. Comparison of posterior skin, as well as epidermis protected from sunlight, Serial Analysis of Gene
Expression (SAGE) was performed. A SAGE library containing tag recombinants derived from multiple mRNAs was isolated from human post-auricle and pre-auricle skin, and polyA (isolated from nick biopsy samples of the epidermis). ⁺ ) RNA. The sequence of 5,330 cDNA tags derived from mRNA obtained from the posterior SAGE library was determined and these tag sequences were analyzed from the anterior SAGE library 5,105 analyzed. C identified from individual tags
It was compared to the DNA sequence. Represented in both libraries,
Out of a total of 4,742 different tags, 35 tags were found in which the abundance of tags between libraries differed at least 4-fold. Among the mRNAs with altered steady-state levels in sun-damaged skin, those encoding keratin 1, macrophage inhibitory factor, and calmodulin-like skin protein were detected. In addition, the cDNA sequences identified in the SAGE library obtained from epidermal biopsy samples (5,257 cDNA tags) and the SAGE library obtained from full-thickness skin samples. CDNA
A comparison of both sequences showed that after exposure to sunlight, keratinocytes in the epidermis expressed a number of genes with altered steady-state transcript levels. This result suggests a major role of the epidermis in the pathogenic mechanism of large changes in the dermis in chronically sun-exposed skin.

Gene Array Establishment Protocol for Tag Sequencing: ABI Prism 3
10 Genetic Analyzer, Perki
n Elmer, Applied Biosystem
Sequence analysis of the resulting ditag concatemers was performed using s, Shelton, CT. This system is D
It is an automatic device capable of determining the base sequence, or size and amount of NA fragments. It uses polyacrylamide capillary electrophoresis in combination with a multicolor fluorescent DNA detection method.

BigDye Terminat to be used
or Cycle Sequencing Ready
Reaction Kit (PE Cat. # 403
044) Analyzer, Perkin Elme
r, Applied Biosystems, Shel
Ton, CT relies on the so-called thermal cycle sequencing method, ie, the procedure of Sanger's fluorescent dideoxy sequencing method, in combination with the linear amplification method of DNA templates.

As a typical example, one sequence-insert size check PCR reagent (c
oncatamer-insert-size-che
ckPCR reaction) 1 μl (about 100n
g) to the Ready reaction Mix (Am
plitaq FS, sequencing buffer (se
quenching buffer), and fluorescently labeled ddNTP) 4 μl, M13 Reverse pr)
imager 3.2 pmol, and ddH ₂ O 11 μl
Was added to a 200 μl PCR tube containing This solution was mixed and mixed with ABI GeneAmp PCR System 9
700, Perkin Elmer, Applied
Cycling sequencing reactions were performed on Biosystems, Shelton, CT under the following conditions: 25 cycles (96 ° C. for 10 seconds, 55 ° C. for 5 seconds, 60 ° C. for 4 minutes).

The resulting extended product was purified by adding 2 μl of 3M sodium acetate and 50 μl of 95% ethanol to the tube. After at least 15 minutes precipitation, the mixture was spun at maximum speed for 20 minutes, the supernatant carefully aspirated, and the remaining pellet washed twice with 70% ethanol. The pellets are dried, and Templ
ate Suppression reagent (P
E Cat. # 401674) Redissolved in 20 μl. Combine up to 96 of these tubes into an ABI P
rism 310 Genetic Analyzer
Charged into.

Conversion of Tag to Gene The 10 to 11 base pairs constituting the SAGE tag are converted into NCBI.
SAGE database (http://www.nc)
bi. nlm. nih. Gov / SAGE /). The tag option for the genetic map was selected.
This identified UniGene clusters that matched this tag. A sequence matching this ID was also identified. The individual sequences containing this tag are then labeled with NCBI.
Extracted from Multiple Sequence A
The lightment is complete. Reverse transcription of the longest clone then revealed a putative primary structure of the protein.

Northern blot analysis: To confirm the altered expression in the photodamage shown by SAGE, three different messages were used to confirm (confirm).
(mattery) Northern blot was performed. This is shown in Table A below. These are in good agreement with the relationships determined by SAGE. Keratin 1 increased 7-fold,
MIF, or migration inhibitory factor, increased 4-fold and hrp S
9 (human ribosomal protein S9) increased 2.2-fold.

Keratin 1, macrophage migration inhibitory factor (MIF), and human ribosomal protein S9 (h
In poly (A ⁺ ) RNA obtained from the same anterior and posterior skin samples used to construct the SAGE library, using radiolabeled cDNA complementary to the mRNA for rp S9). These mR
The level of NA was determined. Control GAPDH
keratin 1 mRNA, MIF mRNA, in the skin of the anterior and posterior ear compared to the level of mRNA,
And the levels of hrp S9 (Table A below) were consistent with our SAGE tag recovery data.

[0061]

[Table 3]

Example 2 The following example provides data for tag sequencing.
AGE analysis. SAGE analysis was performed as described in Velculescu et al. 1995. In essence,
Double-stranded cDNA was synthesized from mRNA using a biotinylated oligo dT primer and then digested with Nla III. This biotinylated 3'most cDNA fragment was treated with streptavidin magnetic beads (Dynal, O).
slo, Norway) and divided into two different aliquots. Bsmf I recognition site, Nla III
Recognition site and PCR primer binding (primin
g) Two different oligonucleotide linkers containing sites were attached to the DNA in each sample. Bsmf
After digestion with I, the tag is ligated and the ditag product is
It was isolated by CR amplification and digestion with Nla III, concatenated, and continuously pZero vector (I
nvitrogen, Carlsbad CA). Individual bacterial colonies containing recombinant plasmid were treated with M13 forward primer and M13 forward.
The insert size was checked by PCR using reverse primers. Then at least 4
The PCR product derived from the 00 bp insert was
igDye Terminator Kit (Perk
in Elmer, Foster City, CA) and a 310 ABI automated DNA sequencer. The tags were compared to the Genbank / EMBL database and the linker-derived duplicates (d
The SAGE 2000 software program (ver) to identify and exclude tags
Sequences were analyzed according to section 4.12). The differentially expressed mRNA-derived tag was labeled with NCBI's S
AGE map "tag to gene (tag to
ne) ”software was used for further analysis.

SAGE Library Using poly (A ⁺ ) RNA isolated from nick biopsies of pre- and post-auricular skin and pooled epidermis from one donor, three SAGE libraries were used. Different SAGE libraries were constructed. Excluding the linker-derived tag, 4,830 S derived from the skin of the anterior auricle
AGE tag, and derived from the skin behind the auricle 4,
990 tags were produced. In addition, 5,215 SAGE tags derived from human epidermis were produced.
Putting these together, the 15,035 tags are
It represented 6,598 specific genes.

Analysis of SAGE tags obtained from the skin behind the auricle Among the 2,858 specific tags obtained from the skin behind the auricle, 127 tags were observed at least 5 times, and their repeatability was observed. The total number of tags in the group represented 32% of the total number of tags. 2,254 out of the few remaining tags
This tag was detected only once. Table III lists the 50 most abundant posterior SAGE tags detected,
The frequencies of these tags, reliable matches with UniGene, and corresponding GenBank accession numbers are listed. The tags derived from mitochondrial DNA were excluded. Wherever possible, the 15th in the SAGE tag sequence (CATG + 11 bp) was used to distinguish multiple matches for the same tag. All but two (tag 13 and tag 33) were assigned to at least one gene. Tag 33 (ACCTCCACTG) is Uni
Tag 13 (ACTTTTTT) was assigned only to the EST cluster in the Gene database.
CAA) did not reliably match to any UniGene cluster. In addition, NCBI SA
According to the GE database, only tag 33 was found in low copy number in other SAGE libraries produced from primary ovarian tumors. Twelve tags were multiply assigned; five of these tags matched sequences derived from two different genes and seven were from two or more different genes. Many of these high-abundance tags are known to be widely expressed in various cell types, particularly genes encoding ribosomal proteins, genes involved in protein synthesis (elongation factor 1), cytoskeletal genes. (Lamin A / C), and a gene active in energy metabolism (glucose phosphate isomerase). MRNA derived from a gene known to be specifically expressed in skin
A tag that conforms to was also found. Among these most abundant SAGE tags specific to skin, some keratins, as well as galectin-7 and calgranulin (c
alganulin), a tag derived from the mRNA coding for all (generally found in all layers of skin) was detected.

[0065]

[Table 4]

Analysis of SAGE Tags Obtained from Pre-Auricular Skin Of the 4,830 tags produced from pre-auricular skin, 2,931 were found to be specific. Of these, 127 specific tags (4%) occurred more than 5 times; 30% of the total amount of tags was represented by these repetitive tag sequences. Almost 50 out of all tags
%, Or 2,393, appeared only once.
Table 2 shows the S of ours composed of the skin of the anterior part of the auricle.
Figure 5 shows a list of the 50 most abundant tags detected in the AGE library. 1 for the skin behind the pinna
All tags (ACCTCCCACTG, tag 22 in anterior skin, tag 33 in posterior skin) are compatible with at least one gene, and multiple tags are associated with more than one gene. I was able to assign it. It was also found that almost all of the most abundant preauricular skin tags were high copy number in the posterior auricular skin. With the exception of tag 28 (ATCCGCGAGGC, calmodulin-like skin protein), all the tags in the list of the 50 most abundant preauricular tags are among the 50 most abundant tags in the posterior auricle. It was found to be present or detected with almost the same number of tags. Most of the most abundant tags in the anterior skin were derived from the mRNA encoded by the housekeeping genes and were used in previous SAGE studies with other tissues (Chen et al., 1998; Velculescu and others,
1997).

[0067]

[Table 5]

Analysis of SAGE Tags Obtained from Epidermis 5,215 SAGE tags were generated from epidermal nick biopsies, which corresponded to 2,982 specific genes. The tag distribution in this library was similar to that observed in the SAGE library obtained from the anterior and posterior skin libraries. The most abundant tag in the epidermis library (5 or more tags) is
It corresponded to 4% of the specific tags. The single tag represented 80% of the specific tags and 45% of all the tags present in this SAGE library. The 50 most frequent epidermal tags are listed in Table V. Of these 50 tags, 2 did not show any gene matches when compared to the UniGene database, 3 tags were only assigned to the UniGene EST cluster and 14 tags (28 %) Did not belong to a single gene. Of the 7 most highly expressed genes, 4 different keratins (keratins 1, 10,
5 and 14) were generally found to be expressed in epidermal keratinocytes. The genes for the intermediate filament protein keratins 5 and 14 are known to be highly expressed in the basal layer of the epidermis, whereas keratins 1 and 10 are mainly found in the epidermal basal epithelium.
asal layer). Filaggrin that cross-links keratin, and also cross-links of intermediate filaments (cross-li)
50), and tags derived from mRNA encoding plakoglobin, which is
It was among the most abundant tags. The rest of these tags in this table are widely derived from genes known to be expressed in many different tissues.

[0069]

[Table 6]

Example 3 This SAG of mRNA profile in human skin
E-analysis identified more than 15,000 tags, which corresponded to mRNA from more than 6,500 different genes. Isolated from chronically sun exposed anterior skin and sun protected posterior skin from a patient suffering from sun damage undergoing selective facial plastic surgery In poly (A ⁺ ) RNA, these mRNAs were identified in full-thickness skin. By choosing this model of pre- and post-auricular skin, at least some of the limitations of other model systems, including the inherent problems of mice for studying solar elastic fibrosis, are addressed. It was avoided. The use of human skin exposed to sunlight also made it possible to study the effects of natural sunlight, rather than identifying the effects of different components of sunlight. Brown et al. (Brown et al., 20
Recent work by (00), for example, reports that ordinary fluorescent sunlamps are unsuitable for replacing natural sunlight. Moreover, cell culture models rarely adequately represent different cell types, as well as the three-dimensional structure of tissue, as a result of, for example, neglecting interactions between cell types. In addition, skin biopsies in this study were taken from adjacent areas of the face in an attempt to minimize phenotypic differences between different areas of the body on the skin. But,
The use of human skin in the anterior and posterior auricles limits the possibility of controlling or affecting experimental conditions such as total time of exposure to sunlight. Moreover, our model is a controlled and limited U
It has been found that it does not represent the changes caused by exposure to V light, but reflects changes in the steady-state levels of mRNA over the years of exposure to sunlight.

Although full-thickness human skin contains a variety of different cell populations, the most abundant cell type in human skin is keratinocytes derived from the epidermis. Therefore, it was expected that the mRNA profile obtained from full-thickness skin would broadly reflect the spectrum of mRNA derived from keratinocytes, and this result was actually obtained. The 50 most abundant mRNAs in both the anterior and the posterior skin were identified in a SAGE library obtained from nick biopsies of the epidermis.
When compared to the RNA profile, many of these tags encode mRNAs for proteins commonly found in the epidermis.
It is clearly derived from NA. Examples of these are keratin, galectin-7, calmodulin-like skin proteins.

Similar analysis of tags obtained from the SAGE library of human dermal fibroblasts (data not shown) is expected to be among some of the most abundant in dermal fibroblasts. MRNA was revealed. These mRNAs include pro α1 (I) and pro α2
Included are mRNAs encoding (I), and proα1 (III) collagen, as well as some matrix metalloproteinases. No tags derived from these mRNAs were observed in our full-thickness skin library, which indicates that this SAGE of full-thickness skin was
Supporting the conclusion that most of the abundant mRNA observed in the analysis was derived from epidermal keratinocytes.

Tables I and II list tags derived from 34 different genes with increased or decreased abundance between preauricular and retroauricular skin. This change in tag number directly reflects changes in steady-state levels for mRNAs in which these tags were induced, and indirectly reflects changes in gene expression. This underlying assumption is
That is, a change in mRNA level is reflected in a change in the amount of protein encoded by the mRNA.

Of the 34 different mRNAs represented by at least 4-fold difference in abundance between the anterior and posterior auricle skin, 6 mRNAs encode ribosomal proteins and 2 mRNAs mRNA encodes a translation initiation factor protein. In this study, it is hypothesized that such changes in ribosomal and initiation factor protein mRNAs reflect general changes in protein synthesis associated with chronic exposure to sunlight. Since the tags derived from mRNAs encoding ribosomal proteins are commonly present in most SAGE studies, most authors have not placed special functional importance on the appearance of these tags.

The four tags in Tables I and II were shown to be multi-matched. Three of these tags correspond to stretches of poly (A). Since the SAGE tag is composed of the 3'end of the mRNA, these poly (A) sequences almost certainly represent one or more mRNs.
It corresponds to a stretch of poly (A) within the 3'untranslated region (UTR) of A, or a poly (A) tail added after transcription at the 3'end of the 3'UTR of mRNA. In both cases, tag matching to these common sequences in most mRNAs resulted in the mRNs in which these tags were derived.
More accurate identification of A is hindered. Similarly, hypothetical protein
The UniGene match for the tags in Table II, identified as (AF151075), represents a previously identified EST cluster of unknown function. M predicted to synthesize protein AAB542440
ESTs with limited homology to RNA are also ESTs that encode proteins of unknown function. 2 not identified in GenBank in Table I
Single tag is a mRNA that was not previously identified as EST
Equivalent to.

With respect to the remaining tags, which code for 18 different mRNAs, it is impressive that the proteins coded by these mRNAs correspond to a functionally distinct group of proteins which are mainly restricted to the epidermis. Taken together, these changes suggest protective mechanisms against chronic exposure of the skin, particularly the epidermis, to UV irradiation, including a persistent inflammatory response as indicated by elevated levels of MIF. In addition, IGFBP-6, CLSP, and EFABP (all involved in keratinocyte differentiation)
Indicates an increase in the steady-state level of mRNA, and
Bcl-2 antagonist and Bax inhibitor 1
An increase in the level of apoptosis inhibition by ss means an alteration in the keratinocyte proliferation-differentiation cycle in sun-damaged skin. In addition, Ca ²⁺ levels are
It is not surprising that mRNA levels encoding some Ca ²⁺ binding proteins are also altered in sun-damaged skin, as it is known to be an important factor for this cycle of switching in keratinocytes.

The pre- and post-auricular SAGE libraries described herein consisted of skin samples obtained from a 55 year old female donor who was then undergoing selective facial plastic surgery. Therefore, this anterior skin sample represented skin that had been repeatedly exposed to sunlight for decades. To date, few studies have addressed the biosynthetic consequences of chronic and repeated exposure to sunlight. For example, Voorhees et al., UV-induced, MAP kinase of matrix metalloproteinases as the underlying mechanism for aberrant remodeling of collagen and other components of connective tissue of the dermis during repeated exposure to sunlight. Proposes an attractive hypothesis of activation mediated by.

In summary, the SAGE analysis performed in accordance with the present invention is the first attempt to obtain a comprehensive profile of biosynthetic changes in human full-thickness skin with chronic exposure to sunlight. is there. A total of 6,500 transcripts analyzed identified 18 different mRNAs with significantly altered steady-state levels associated with chronic exposure to sunlight.

Although the present invention has been described herein, more particularly, with reference to certain preferred embodiments thereof,
Those skilled in the art will appreciate the numerous possible variations, modifications and alternatives that fall within the scope and spirit of the invention as described above.
All of these modifications and variations are within the scope of the invention as described and claimed herein, the invention being limited only by the appended claims, which are reasonable. Shall be broadly construed. Throughout this application, various publications have been referenced. The entirety of each of these publications is incorporated herein by reference.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Kurt Machine Schilling             United States, New Jersey             07020, Edgewater, River Law             De 45, Unilever Research You             S Incorporated (72) Inventor, Charles Boyd             Honolulu, Hawaii 96822, USA             Le, Suite 280, Utsudrone Dora             Eve 2800, University of Hawa             I (72) Inventor Johann Woo-atsutsu             Honolulu, Hawaii 96822, USA             Le, Suite 280, Utsudrone Dora             Eve 2800, University of Hawa             I F-term (reference) 4B024 AA11 CA04 CA12 HA12                 4B063 QA19 QA20 QQ08 QQ53 QR08                       QR42 QR56 QS25 QS34 QX02

Claims (6)

[Claims] 1. A personal care method for detecting an optical disorder, comprising: (a) SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 6
0, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 6
7, using at least one marker of photodamage, selected from one or more sequences selected from the group consisting of SEQ ID NO: 68, and SEQ ID NO: 69;
(B) A method comprising detecting a change in the marker to determine the presence of a photodamage. 2. The detecting step (b) further comprises the step of (b1) comparing the first skin sample with a second skin sample to determine if there is a change in the marker. The method described. 3. A personal care method for measuring the skin condition in a sample of epidermis or dermis or whole skin, which comprises (a) measuring the first gene expression of a predetermined skin sample having a known skin condition. Steps,
(B) measuring the second gene expression of a predetermined skin sample having no known skin condition, and (c) the first.
Identifying a plurality of markers that identify a known skin condition of changes in gene expression of the second gene expression of the second gene. 4. The identifying step (c) further comprises the step of: (c1) determining a marker by assessing a change in gene expression between the first gene expression and the second gene expression. The method described in. 5. The method of claim 3, wherein the known skin condition is selected from conditions including photodamage, aging, dry skin, and oily skin. 6. The known skin condition is photodamage, and a plurality of markers are SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 5.
3, sequence number 54, sequence number 55, sequence number 56, sequence number 57, sequence number 58, sequence number 59, sequence number 6
0, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 6
The method of claim 3, selected from the group consisting of 7, SEQ ID NO: 68, and SEQ ID NO: 69.